Activatable prodrug for controlled release of an antimicrobial peptide via the proteases overexpressed in Candida albicans and Porphyromonas gingivalis

Candida albicans and Porphyromonas gingivalis are prevalent in the subgingival area where the frequency of fungal colonization increases with periodontal disease. Candida's transition to a pathogenic state and its interaction with P. gingivalis exacerbate periodontal disease severity. However, current treatments for these infections differ, and combined therapy remains unexplored. This work is based on an antimicrobial peptide that is therapeutic and induces a color change in a nanoparticle reporter. Methods: We built and characterized two enzyme-activatable prodrugs to treat and detect C. albicans and P. gingivalis via the controlled release of the antimicrobial peptide. The zwitterionic prodrug quenches the antimicrobial peptide's activity until activation by a protease inherent to the pathogens (SAP9 for C. albicans and RgpB for P. gingivalis). The toxicity of the intact prodrugs was evaluated against fungal, bacterial, and mammalian cells. Therapeutic efficacy was assessed through microscopy, disk diffusion, and viability assays, comparing the prodrug to the antimicrobial peptide alone. Finally, we developed a colorimetric detection system based on the aggregation of plasmonic nanoparticles. Results: The intact prodrugs showed negligible toxicity to cells absent a protease trigger. The therapeutic impact of the prodrugs was comparable to that of the antimicrobial peptide alone, with a minimum inhibitory concentration of 3.1 - 16 µg/mL. The enzymatic detection system returned a detection limit of 10 nM with gold nanoparticles and 3 nM with silver nanoparticles. Conclusion: This approach offers a convenient and selective protease sensing and protease-induced treatment mechanism based on bioinspired antimicrobial peptides.


I. Experimental Procedures
Peptide synthesis.Peptides were synthesized as prepared in previous work [1].Briefly, an automated Eclipse™ peptide synthesizer (AAPPTec, Louisville, KY) was utilized for standard solid phase Fmoc synthesis on Rink-amide resin (0.55 mmol/g, 200 mg).Amino acids were coupled (C to N) under nitrogen protection with 0.2 M Fmoc-amino acid (5 equiv.) in 3 mL DMF, 0.2 M HBTU (5 equiv.) in 3 mL DMF, 0.4 M DIPEA (7.5 equiv.) in 3 mL DMF, and 20% (v/v) piperidine in 2 x 4 mL DMF for each coupling cycle.The resulting resin and peptide were then transferred to a syringe filter (Torviq Inc.) and washed with three rounds of DMF (5 mL each) and three rounds of DCM (4 mL each).It was then dried under vacuum.For acetylated peptides, the N-terminal was acetylated using the following recipe: 4 mL of DMF, 0.5 mL of Pyridine, and 0.5 mL of acetic anhydride.The solution was then subjected to light stirred for 30 minutes before being purged and washed with DMF and DCM as previously mentioned.The dried peptides were next cleaved from the resin using a 5 mL cocktail solution that consists of: 83% TFA, 5% H2O, 5% thioanisole, 5% phenol, and 2% µL EDDET.The incubated solution was gently rotated for 2 hours.The resin was then filtered and the filtrate containing the crude peptide was collected and precipitated using cold ethyl ether (20 mL, -20 ºC) and centrifuged three times (8,000 rpm, 3 minutes).Once the supernatant was removed, the precipitated pellets were dried and resuspended using 10 mL of ACN/H2O mixtures wherein the percentage of ACN was controlled based on the solubility of the peptide.
Peptide purification and characterization.Peptide purification was conducted as done by Retout et al [1].Once dry, the crude peptides were purified with a Shimadzu LC-40 HPLC system equipped with a LC-40D solvent delivery module, photodiode array detector SPD-M40, and degassing unit DGU-403.An injection of 2 mL was utilized with a Zorbax 300 BS, C18 column (5 µM, 9.4 × 250 mm) using an elution flow rate of 5 mL/ min over a 40-minute gradient from 10% to 95% acetonitrile in water (0.05% TFA).The peptide bond absorbance of 220 nm was monitored closely, and the elution was collected for characterization.Electrospray ionization mass spectrometry (ESI-MS) on the positive ion mode via the Micromass Quattro Ultima mass spectrometer provided by the Molecular MS Facility (MMSF) at UC San Diego was utilized with an MEOH/ H2O mixture (1:1, v/v) and an injection volume of 5 µL.Some compounds were characterized using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer using a linear positive mode.Here, a-Cyano-4-hydroxycinnamic acid (HCCA) was the matrix utilized at a ratio of 1:3 and 2 µL was placed and dried with a heat gun to be analyzed.
Duplicates were used as recommended by the MMSF.Fractions containing the pure peptide as confirmed by electrospray ionization mass spectrometry (ESI-MS, positive ion mode) were lyophilized in a FreeZone Plus 2.5 freeze dry system (Labconco Corp., Kansas, MO) and aliquoted and stored in dry conditions at 2 ºC for further use [2].
Michaelis-Menten kinetics.The peptide substrate was resynthesized with acetylated lysine residues and linked to cyanine-NHS ester dyes (Cy5.5 and Cy3) using the amine groups in the form of NH3 + and lysine at the N-terminal and the C-terminal, respectively: (V[aK][aK][aK]DVVDK). Acetylated lysine residues were employed to guide the dye to the lysine on the end terminal.Herein, 0.5 mg of the peptide dissolved in 369 µL of anhydrous DMSO with 31 µL of 1% (v/v) triethylamine (2.25 µmol, 1.5 equiv.)Next, Cy5.5 and Cy3 were added to the solution (3.5 µg, 4.50 µmol) and the solution was covered with aluminum foil and left to stir overnight at 300 rpm.The crude reaction was then dried under vacuum centrifugation using a Vacufuge Plus (Eppendorf, Hamburg) at 60 ºC until light-reflecting pellet was formed.The pellet was then resuspended in 25% ACN/ H2O (v/v) and separated using HPLC.
The heterodimer was diluted in 9.5 mM MES, 2.7 mM KCl, 140 mM NaCl (pH 5.5) buffer to reach a final [S] and distributed in 24 wells within a 96-well plate for duplicate measurements of each concentration.The enzyme ([E]0 = 200 nM with respect to a final 100 μL volume) was then added to each well.The plate was incubated at 37 °C and the fluorescence intensity (684 nm for Cy5.5 and 570 nm for Cy3) was recorded over 12 h with 1 min intervals between each cycle.Measurements were performed in duplicates.The signal values at 30 min readout time were averaged and plotted against substrate concentrations; error bars represent the standard error of the means.The ∆PL = PL30 min -PL0 min was then correlated to product concentration using a standard curve: ∆PLCy5.5 vs. [fully-digested FRET probe].Data were then fitted to the following Michaelis-Menten equation: .
PEP-FOLD Simulation.PEP-FOLD is a computational tool used for predicting protein structure, specifically focusing on predicting the three-dimensional (3D) structure of peptides or small proteins [3,4].PEP-FOLD operates based on a de novo approach to structure prediction.The amino acid sequences were provided.PEP-FOLD then employs a physics-based energy function to calculate the energetically favorable conformations of the peptide chain in water.This involves considering various forces and interactions, such as bond angles, dihedral angles, and nonbonded interactions.PEP-FOLD generates a large number of potential conformations or structures for the given peptide sequence.This is often done using a Monte Carlo or molecular dynamics sampling approach.Each generated conformation is assigned a score based on its energy and the agreement with experimental or theoretical constraints.The algorithm selects the most energetically favorable conformations as potential predictions for the 3D structure of the peptide.Finally, PEP-FOLD provides the user with the predicted 3D structure(s) of the input peptide sequence.It is important to note that the accuracy of structure prediction tools can vary depending on the length and complexity of the peptide sequence.
Confocal Microscopy.Mammalian cells were seeded at a density of 1×10 Next, 20 µL of combined H2O2 Substrate (Promega) and test compound to cells and mixed for a final well volume of 100 µL and the final H2O2 Substrate concentration of 25µM.The plate was then incubated for 4 hours at 37 °C before 100µl of ROS-Glo™ Detection Solution was added to each well.Finally, the plate was incubated for 20 minutes at room temperature and the relative luminescence was recorded using a plate reader (Synergy H1, BioTek).

Limit of Detection.
The limit of detection (LoD) was calculated using the limit of blank (LoB) as demonstrated by Armbruster et al [5].The LoB uses a blank measurement to define the highest signal generated from the sample with no analyte.LoB was calculated using the mean (meanblank) and standard deviation (SDblank) of a blank sample: LoB = meanblank + 1.645 (SDblank) Based on this, the LoD is defined as the lowest analyte concentration that can be differentiated from the LoB.Here, the LoD represents an analyte concentration at which 95% of measured samples are readily differentiated from the LoB while the remaining 5% can contain no analyte: LoD = LoB + 1.645 (SDlow concentration sample).Statistical Analysis.Statistical analysis was conducted using GraphPad Prism 10.Percent toxicity was quantified by averaging the absorbance for each repetition, and error bars represent the standard deviation across eight wells.For intra-and inter-assay variability, data from two consecutive experiments were analyzed.Percent total variation was calculated using ANOVA to compare viability before and after cleavage by recombinant prodrug.A P-value of < 0.05 was considered significant.The null hypotheses were: (1) There is no significant difference in viability among cell types.(2) There is no significant difference in viability among treatments.(3) There is no interaction effect between cell type and treatment on viability.Multiple Unpaired Student's t tests were also performed to compare the significance between variables.A two-way ANOVA was performed with the cell type and treatment as independent variables and viability as the dependent variable.The null hypotheses are: (1) There is no significant difference in viability among cell types and (2) There is no significant difference in viability among treatments.(3) There is no interaction effect between cell type and treatment on viability.The calculated P-value for all three was <0.0001 suggesting significant data and as such (P < 0.05), a statistically significant difference in viability between cell types, a difference in viability and an effect between cell type and treatment.Eight replicates were used which resulted in an average statistical power of 0.9 when α = 0.05.Thus, there is a strong chance of correctly rejecting the null hypothesis if there is a true effect under these experimental parameters.

Figure S6 .Figure
Figure S6.RgpB cleavable substrate FRET probe characterization.(A) RgpB cleavable substrate peptide structure: Ala -Gly -Pro -Arg -Ile -Asp -Lys.ESI-MS presenting two clear peaks at [M+2H] 2+ = 378.62 and [M+1H] 1+ = 756.10.(B) Structure of RgpB cleavable probe with conjugated NHS-dyes on either terminal with theoretical mass 1513.2932Da. (C) ESI-MS spectra with clear peak at [M+2H] 2+ = 505.26confirming successful conjugation.(D) Michaelis-Mentenmaster curve describing the rate of reaction with an increase in substrate concentration to determine kcat/ KM, the second-order rate constant reaction rate of the enzyme-substrate complex to product.Here, a higher ratio suggests a higher rate of conversion.Inset parameters present: [E]active, the active enzyme concentration used, 200 nM.Catalytic constant, kcat, the number of probe molecules converted by enzyme per second.The Michaelis-Menten constant, KM, an inverse measurement of affinity.

Figure S8 .
Figure S8.Microorganism growth for disk diffusion test.(A) String of pearls" characteristic of C. albicans formation.(B) P. gingivalis colonies with characteristic black pigmentation plated and grown anaerobically on blood agar.

Figure S11 .Figure S12 .Figure S13 .Figure S14 .Figure S15 .S1Figure S16 .
Figure S11.Confocal microscopy of mammalian cells.HEK293T demonstrating a deconstructed cell membrane when treated with bleach (positive control) intact cell membrane after treatment with prodrugs S1 and G1 for 3 hours at 37 °C.Cells were stained at a final concentration of 1X at 37 °C for 15 minutes with CellBrite Fix Membrane Stain 640, fixed with 4% paraformaldehyde, and imaged with a Leica SP8 with lighting deconvolution at an excitation/ emission wavelength of 638 nm/ 667 nm.

Figure S17 .
Figure S17.Statistical analysis to highlight the antimicrobial specificity of proteaseactivated prodrugs.A two-way ANOVA was performed with the cell type and treatment as independent variables and viability as the dependent variable.The null hypotheses are: (1) There is no significant difference in viability among cell types and (2) There is no significant difference in viability among treatments.(3) There is no interaction effect between cell type and treatment on viability.The calculated P-value for all three was <0.0001 suggesting significant data and as such (P < 0.05), a statistically significant difference in viability between cell types, a difference in viability and an effect between cell type and treatment.Eight replicates were used which resulted in an average statistical power of 0.9 when α = 0.05.Thus, there is a strong chance of correctly rejecting the null hypothesis if there is a true effect under these experimental parameters.

Figure S19 .
Figure S19.C. albicans TEM images of cellular morphology at increased magnification.Top: Untreated cells showing intact cell membranes at 5 µm and 2 µm scale bars (left to right).Bottom: Treated cells with S1 for 3 hours at 37 °C showing deconstructed cell structure and no evident cell membrane or organism.

Figure S20 .
Figure S20.P. gingivalis TEM images of cellular morphology at increased magnification.Top: Untreated cells showing intact cell membranes at 10 µm and 2 µm scale bars (left to right).Bottom: Treated cells with G1 for 3 hours at 37 °C showing notable decrease in cell density and destruction of cell structure.

Figure S21 .Figure S22 .
Figure S21.Intact prodrug effects on membrane integrity.(A, B) C. albicans TEM images of cellular morphology after treatment with G1 for 3 hours at 37 °C; there are intact cell structures and cell membranes.Panel A scale bar is 10 µm and B is 1 µm.(C, D) P. gingivalis TEM images of cellular morphology after treatment with S1 for 3 hours at 37 °C showing intact cell structures and membranes.Panel C scale bar is 2 µm and D is 1 µm.
Proteases secreted by the microorganisms that cleave an aspartic acid substrate bond.